Transcript Industrial Biotechnology

Industrial Biotechnology
Lecturer Dr. Kamal E. M. Elkahlout
Assistant Prof. of Biotechnology
1
CHAPTER 4
Industrial Media and Nutrition of Industrial
Organisms
2
• It is important to use good, adequate, & industrially
usable medium.
• Enhances harness of the organism’s full industrial
potentials.
• If media was not suitable, the production of the
desired product will be reduced & toxic materials
may be produced.
• Liquid media are generally employed in industry
because they require less space.
• LM are more amenable to engineering processes,
and eliminate the cost of providing agar and other
solid agents.
THE BASIC NUTRIENT REQUIREMENTS OF
INDUSTRIAL MEDIA
• For industrial or for laboratory purposes, media
must satisfy the needs of C, N, minerals, growth
factors, and water.
• No inhibitory materials.
• Complete analysis of the organism’s nutrients needs
should be performed.
• C or energy requirements are usually met from
carbohydrates (glucose, starch or cellulose & ….,
etc) .
• Energy sources may include hydrocarbons, alcohols,
or even organic acids.
• In formulation industrial medium, the carbon
content must be adequate for the production of
cells.
• For most organisms the weight of organism
produced from a given weight of carbohydrates
(known as the yield constant) under aerobic
conditions is about 0.5 gm of dry cells per gram of
glucose.
• Carbohydrates are at least twice the expected
weight of the cells and must be put as glucose or its
equivalent compound.
•
•
•
•
•
•
•
•
•
Nitrogen is a key element in the cell.
Most cells would use ammonia or other nitrogen salts.
For bacteria the average N content is 12.5%.
To produce 5 gm of bacterial cells per liter would
require about 625 mg N (Table 4.1).
Any nitrogen compound which the organism cannot
synthesize must be added.
Minerals form component portions of some enzymes.
The major minerals needed include P, S, Mg and Fe.
Trace elements: manganese, boron, zinc, copper and
molybdenum.
Growth factors include vitamins, amino acids and
nucleotides and must be added to the medium if the
organism cannot manufacture them.
CRITERIA FOR THE CHOICE OF RAW MATERIALS USED
IN INDUSTRIAL MEDIA
•
•
•
•
•
•
•
•
Cost of the material
Ready availability of the raw material
Transportation costs
Ease of disposal of wastes resulting from the raw
materials
Uniformity in the quality of the raw material and
ease of standardization
Adequate chemical composition of medium
Presence of relevant precursors
Satisfaction of growth and production requirements
of the microorganisms
• Cost of the material
• The cheaper the raw materials the more
competitive the selling price of the final product.
• Lactose is more suitable than glucose in some
processes (e.g. penicillin production) because of the
slow rate of its utilization, it is usually replaced by
the cheaper glucose.
• The raw materials used in many industrial media
are usually waste products from other processes.
• E.g., Corn steep liquor and molasses.
• Ready availability of the raw material
• If it is seasonal or imported, then it must be
possible to store it for a reasonable period.
• The material must be capable of long-term storage
without change in quality.
• Transportation costs
• The closer the source of the raw material to the
point of use the more suitable it is for use, if all
other conditions are satisfactory.
• Ease of disposal of wastes resulting from the raw
materials
• The disposal of industrial waste is rigidly controlled
in many countries.
• Waste materials often find use as raw materials for
other industries.
• Thus, spent grains from breweries can be used as
animal feed.
• But in some cases no further use may be found for
the waste from an industry.
• Its disposal could be expensive.
• When choosing a raw material therefore the cost, if
any, of treating its waste must be considered.
• Uniformity in the quality of the raw material and ease
of standardization
• Composition must be reasonably constant in order to
ensure uniformity of quality in the final product and the
satisfaction of the customer.
• E.g., molasses as waste product of sugar industry.
• Each batch of molasses must be chemically analyzed
before being used in a fermentation industry in order to
ascertain how much of the various nutrients must be
added.
• A raw material with extremes of variability in quality is
undesirable as extra costs are needed.
• - Analysis of the raw material,
• - Nutrients which may need to be added to attain the
usual and expected quality in the medium.
• Adequate chemical composition of medium
• The medium must have adequate amounts of C, N,
minerals and vitamins in the appropriate quantities and
proportions necessary for the optimum production of
the commodity in question.
• The compounds in the medium must utilizable by the
organisms.
• Thus most yeasts utilize hexose sugars, whereas only a
few will utilize lactose.
• Cellulose is not easily used and is utilized only by a
limited number of organisms.
• Some organisms grow better in one or the other
substrate.
• Fungi will for instance readily grow in corn steep liquor
while actinomycetes will grow more readily on soya
bean cake.
• Presence of relevant precursors
• Precursors necessary for the synthesis of the finished
product.
• Precursors often stimulate production of secondary
metabolites either by
• - increasing the amount of a limiting metabolite,
• - by inducing a biosynthetic enzyme or both.
• Precursors include amino acids & small molecules.
• For penicillin G to be produced the medium must
contain a phenyl compound.
• Corn steep liquor contains phenyl precursors.
• Other precursors are cobalt in media for Vitamin B12
production & chlorine for the chlorine containing
antibiotics, chlortetracycline, & griseofulvin (Fig. 4.1).
• Satisfaction of growth and production requirements of
the microorganisms
• Many industrial organisms have two phases of growth
in batch cultivation: the phase of growth, or the
trophophase, and the phase of production, or the
idiophase.
• In the first phase cell multiplication takes place rapidly,
with little or no production of the desired material.
• It is in the second phase that production of the material
takes place, usually with no cell multiplication and
following the elaboration of new enzymes.
• Often these two phases require different nutrients or
different proportions of the same nutrients.
• The medium must be complete and be able to cater
for these requirements.
• For example high levels of glucose and phosphate
inhibit the onset of the idiophase in the production
of a number of secondary metabolites of industrial
importance.
• The levels of the components added must be such
that they do not adversely affect production.
SOME RAW MATERIALS USED IN
COMPOUNDING INDUSTRIAL MEDIA
•
•
•
•
•
•
•
Corn steep liquor
Pharmamedia
Distillers soluble
Soya bean meal
Molasses
Sulfite liquor
Other Substrates (alcohol, acetic acid, methanol,
methane, and fractions of crude petroleum)
Corn steep liquor
• This is a by-product of starch manufacture
from maize.
• As a nutrient for most industrial organisms
corn steep liquor is considered adequate,
• rich in carbohydrates, nitrogen, vitamins, and
minerals.
• highly acidic, it must be neutralized (usually
with CaCO3) before use.
Approximate composition of corn steep
liquor (%)
Pharmamedia
• Yellow fine powder made from cotton-seed
embryo.
• It is used in the manufacture of tetracycline
and some semi-synthetic penicillins.
• rich in protein, (56% w/v) and contains 24%
carbohydrate, 5% oil, and 4% ash
• rich in calcium, iron, chloride, phosphorous,
and sulfate.
Distillers soluble
• By-product of the distillation of alcohol from
fermented grain. (maize or barley)
• It is rich in nitrogen, minerals, and growth
factors.
Composition of maize distillers soluble
Soya bean meal
• The seeds are heated before being extracted for oil that is
used for food, as an antifoam in industrial fermentations, or
used for the manufacture of margarine.
• The resulting dried material, soya bean meal, has about 11%
nitrogen, and 30% carbohydrate and may be used as animal
feed.
• Its nitrogen is more complex than that found in corn steep
liquor
• Not readily available to most microorganisms, except
Actinomycetes.
• It is used particularly in tetracycline and streptomycin
fermentations.
Molasses
• Molasses
• It is a by-product of the sugar industry.
• For the production of cells the variability in molasses
quality is not critical.
• For metabolites such as citric acid, it is very important as
minor components of the molasses may affect the
production of these metabolites.
• High test’ molasses (inverted molasses) is a brown thick
syrup liquid used in the distilling industry and containing
about 75% total sugars (sucrose and reducing sugars) and
about 18% moisture.
• Indeed it is invert sugar, (i.e reducing sugars
resulting from sucrose hydrolysis).
• Produced by hydrolysis of the concentrated juice
with acid.
• In the so-called Cuban method, invertase is used for
the hydrolysis.
• Sometimes ‘A’ sugar may be inverted and mixed
with ‘A’ molasses.
Sulfite Liquor
• Sulfite liquor (also called waste sulfite liquor,) is the aqueous
effluent resulting from the sulfite process for manufacturing
cellulose or pulp from wood.
• During the sulfite process, hemicelluloses hydrolyze and
dissolve to yield the hexose sugars, glucose, mannose,
galactose, fructose and the pentose sugars, xylose, and
arabinsoe.
• Used as a medium for the growth of microorganisms after
being suitably neutralized with CaCO3 and enriched with
ammonium salts or urea, and other nutrients.
• It has been used for the manufacture of yeasts and alcohol.
• Some samples do not contain enough assaimilable
carbonaceous materials for some modern fermentations.
• They are therefore often enriched with malt extract, yeast
autolysate, etc.
GROWTH FACTORS
• Not synthesized by the organism
• Must be added to the medium.
• Function as cofactors of enzymes and may be
vitamins, nucleotides etc.
• The pure forms are usually too expensive for
use in industrial media
• Growth factors are required only in small
amounts.
Some sources of growth factors
• WATER
• Water is a raw material of vital importance in industrial
microbiology.
• Major component of the fermentation medium.
• Cooling, washing and cleaning.
• It is used in large quantities.
• In some industries the quality of the product depends
to some extent on the water.
• To ensure constancy of product quality the water must
be regularly analyzed for minerals, color, pH, etc. and
adjusted as may be necessary.
• Due to the importance of water, in situations where
municipal water supplies are likely to be unreliable,
industries set up their own supplies.
SOME POTENTIAL SOURCES OF COMPONENTS
OF INDUSTRIAL MEDIA
• The materials to be discussed are mostly found in the tropical
countries.
• Any microbiological industries to be sited must use the locally
available substrates.
• Carbohydrate Sources
• Polysaccharides that have to be hydrolyzed to sugar before being
used.
• (a) Cassava (manioc)
• The roots of the cassava-plant Manihot esculenta Crantz (food &
feed) in the tropical world.
• High yielding, little attention when cultivated, and the roots can
keep in the ground for many months without deterioration before
harvest.
• The inner fleshy portion is a rich source of starch and has served,
after hydrolysis, as a carbon source for single cell protein, ethanol.
• In Brazil it is one of the sources of ethanol production.
•
•
•
•
(b) Sweet potato
Ipomca batatas is a warm-climate crop.
It can be grown also in sub tropical regions.
Large number of cultivars vary in the colors of the
tuber flesh and of the skin; they also differ in the
tuber size, time of maturity, yield, and sweetness.
• They are widely grown in the world.
• Regarded as minor sources of carbohydrates in
comparison to wheat, or cassava.
• Do not require much agronomic attention.
• Used as sources of sugar on a semi-commercial basis .
• The fleshy roots contain saccharolytic enzymes.
• The syrup made from boiling the tubers has been used
as a carbohydrate (sugar) source in compounding
industrial media.
• Butyl alcohol, acetone and ethanol have been produced
from such a syrup, and in quantities higher than the
amounts produced from maize syrup of the same
concentration.
• Not widely consumed as food, it is possible that it may
be profitable to grow them for industrial microbiology
media as well as for the starch industry.
• Some variety can yields up to 40 tonnes per hectare, a
much higher yield than cassava or maize.
• (c) Yams
• Yams (Dioscorea spp) are widely consumed in the
tropics. Compared to other tropical
• Cultivation is tedious.
• Enough of this tuber is not produced even for
human food.
• It is inconceivable to suggest for growing solely for
use in compounding industrial media.
• Yams have been employed in producing various
products such as yam flour and yam flakes.
• Mass production may encourage its use or its
wasted peelings as industrial microbiological media.
• (d) Cocoyam
• Cocoyam is a blanket name for several edible members
of the monocotyledonous (single seed-leaf) plant of the
family Araceae (the aroids), the best known two genera
of which are Colocasia (tano) and Xanthosoma (tannia).
• They are grown and eaten all over the tropical world.
• Laborious to cultivate, require large quantities of
moisture and do not store well.
• They are not the main source of carbohydrates in
regions where they are grown.
• Cocoyam starch has been found to be of acceptable
quality for pharmaceutical purposes.
• Should it find use in that area, starchy by-products
could be hydrolyzed to provide components of
industrial microbiological media.
• (e) Millets
• This is a collective name for several cereals whose seeds are
small in comparison with those of maize, sorghum, rice, etc.
• The plants are also generally smaller.
• They are classified as the minor cereals as they generally do
not form major components of human food.
• They are hardy and will tolerate great drought and heat, grow
on poor soil and mature quickly.
• It could become potential sources of cereal for use in
industrial microbiology media.
• Millets are grown all over the world in the tropical and subtropical regions and belong to various genera: Pennisetum
americanum (pearl or bulrush millet), Setaria italica (foxtail
millet), Panicum miliaceum (yard millet), Echinochloa
frumentacea (Japanese yard millet) and Eleusine corcana
(finger millet).
• Millet starch has been hydrolyzed by malting for alcohol
production on an experimental basis as far back as 50
years ago.
• (f) Rice
• Rice, Oryza sativa is one of the leading food corps of the
world, especially in the tropical areas.
• High-cost commodity.
• Ease of mechanization, storability.
• Availability of improved seeds.
• The increase in rice production is expected to become
so efficient for industrial microbiological use.
• Rice is used as brewing adjuncts and has been malted
experimentally for beer brewing.
• (g) Sorghum
• Sorghum, Sorghum bicolor, is the fourth in term of
quantity of production of the world’s cereals, after
wheat, rice, and corn.
• It is used for the production of special beers in
various parts of the world.
• It has been mechanized and has one of the greatest
potential among cereals for use as a source of
carbohydrate in industrial media in regions of the
world where it thrives.
• It has been successfully malted and used in an allsorghum lager beer which compared favorably with
barley lager beer
• (h) Jerusalem artichoke
• Jerusalem artichoke, Helianthus tuberosus, is a
member of the plant family compositae, where the
storage carbohydrate is inulin, a polymer of fructose
into which it can be hydrolyzed.
• It is a root-crop and grows in temperate, semitropical and tropical regions.
Protein Sources
• (a) Peanut (groundnut) meal
• Various leguminous seeds.
• Only peanuts (groundnuts) Arachis hypogea will be
discussed.
• The nuts are rich in liquids and proteins.
• The groundnut cake left after the nuts have been
freed of oil is often used as animal feed.
• Oil from peanuts may be used as anti-foam while
the press-cake could be used for a source of
protein.
• (b) Blood meal
• Blood consists of about 82% water, 0.1%
carbohydrate, 0.6% fat, 16.4% nitrogen, and 0.7%
ash.
• It is a waste product in abattoirs although it is
sometimes used as animal feed.
• Drying is achieved by passing live steam through the
blood until the temperature reaches about 100°C.
• This treatment sterilizes it and also causes it to clot.
• It is then drained, pressed to remove serum, further
dried and ground.
• The resulting blood-meal is chocolate-colored and
contains about 80% protein and small amounts of
ash and lipids.
• (c) Fish Meal
• Fish meal is used for feeding farm animals.
• It is rich in protein (about 65%) and, minerals
(about 21% calcium 8%, and phosphorous 3.5%)
and may therefore be used for industrial
microbiological media production.
• Fish meal is made by drying fish with steam either
aided by vacuum or by simple drying.
• Alternatively hot air may be passed over the fish
placed in revolving drums.
• It is then ground into a fine powder.
• THE USE OF PLANT WASTE MATERIALS IN
INDUSTRIAL MICROBIOLOGY MEDIA:
SACCHARIFICATION OF POLYSACCHARIDES
• Agriculture waste materials and even crops.
• Plentiful and renewable.
• Large amounts of polysaccharides which are in need
for hydrolysis or saccharification to be utilizable by
industrial microorganisms.
• Hydrolyzed polysaccharides may give more available
sugars for microorganisms.
• The sugars could be converted into ethanol for
example or any other commodity produced by Mos.
• Starch
• It is a mixture of two polymers of glucose: amylose
and amylopectin.
• Amylose is a linear (1-4) – D glucan usually having a
degree of polymerization (D.P., i.e. number of
glucose molecules) of about 400 and having a few
branched residues linked with (1-6) linkages.
• Amylopectin is a branched D glucan with
predominantly – D (1-4) linkages and with about 4%
of the – D (1-6) type (Fig. 4.3).
• Amylopectin consists of amylose – like chains of D.
P. 12 – 50.
• Starches differ in their proportion of amylopectin
and amylose according to the source.
• The common type of maize, for example, has about
26% of amylose and 74% of amylopectin.
• Others may have 100% amylopectin and still others
may have 80 – 85% of amylose.
• Saccharification of starch
• Starch occurs in discrete crystalline granules in plants,
and in this form is highly resistant to enzyme action.
• However when heated to about 55°C – 82°C depending
on the
• type, starch gelatinizes and dissolves in water and
becomes subject to attack by various enzymes.
• Before saccharification, the starch or ground cereal is
mixed with water and heated to gelatinize the starch
and expose it to attack by the saccharifying agents.
• The gelatinization temperatures of starch from various
cereals is given in Table 12.1.
• The saccharifying agents used are dilute acids and
enzymes from malt or microorganisms.
• Saccharification of starch with acid
• The starch-containing material to be hydrolyzed is
ground and mixed with dilute hydrochloric acid,
sulfuric acid or even sulfurous acid.
• When sulfurous acid is used itcan be introduced
merely by pumping sulfur dioxide into the mash.
• The concentrations of the mash and the acid, length
of time and temperature of the heating have to be
worked out for each starch source.
• The actual composition of the hydrolysate will
depend on the factors mentioned above.
• Starch concentration is particularly important: if it is
too high, side reactions may occur leading to a
reduction in the yield of sugar.
• At the end of the reaction the acid is neutralized.
•
•
•
•
•
Use of enzymes
Collectively diastase.
They are now called amylases.
Advantages over acids:
(a) since the pH for enzyme hydrolysis is about neutral,
there is no need for special vessels which must stand
the high temperature, pressure, and corrosion of acid
hydrolysis;
• (b) enzymes are more specific and hence there are
fewer side reactions leading therefore to higher yields;
• (c) acid hydrolysis often yields salts which may have to
be removed constantly or periodically thereby
increasing cost;
• (d) it is possible to use higher concentrations of the
substrates with enzymes than with acids.
• Enzymes involved in the hydrolysis of starch
• They are divisible into six groups.
• (i) Enzymes that hydrolyse α – 1, 4 bonds and by-pass α – I, 6
bonding: The typical example is α - amylase.
• This enzyme hydrolyses randomly the inner α - (1 4) - D glucosidic bonds of amylose and amylopectin (Fig. 4.3).
• The cleavage can occur anywhere as long as there are at least six
glucose residues on one side and at least three on the other side
of the bond to be broken.
• The result is a mixture of branched - limit dextrins (i.e., fragments
resistant to hydrolysis and contain the α - D (1-6) linkage (Fig. 4.4)
derived from amylopectin) and linear glucose residues especially
maltohexoses, maltoheptoses and maltotrioses.
• α -Amylases are found in virtually every living cell and the property
and substrate pattern of α - amylases vary according to their
source.
• Animall α - amylases in saliva and pancreatic juice completely
hydrolyze starch to maltose and D-glucose.
• Among microbial α - amylases some can withstand temperatures
near 100°C.
• (ii) Enzymes that hydrolyse the α–1, 4 bonding, but
cannot by-pass the α– 1,6 bonds: Beta amylase: This
was originally found only in plants but has now been
isolated from micro-organisms. Beta amylase
hydrolyses alternate α– 1,4 bonds sequentially from the
non-reducing end (i.e., the end without a hydroxyl
group at the C – 1 position) to yield maltose (Figs. 4.3
and 4.5).
• Beta amylase has different actions on amylose and
amylopectin, because it cannot by-pass the α –1:6 –
branch points in amylopectin.
• Therefore, while amylose is completely hydrolyzed to
maltose, amylopectin is only hydrolyzed to within two
or three glucose units of the α– 1.6 - branch point to
yield maltose and a ‘beta-limit’ dextrin which is the
parent amylopectin with the ends trimmed off.
• Debranching enzymes (see below) are able to open
up the α– 1:6 bonds and thus convert beta-limit
dextrins to yield a mixture of linear chains of
varying lengths; beta amylase then hydrolyzes these
linear chains.
• Those chains with an odd number of glucose
molecules are hydrolyzed to maltose, and one
glucose unit per chain.
• The even numbered residues are completely
hydrolyzed to maltose.
• In practice there is a very large population of chains
and hence one glucose residue is produced for
every two chains present in the original starch.
• (iii) Enzymes that hydrolyze (α —1, 4 and α — 1:6
bonds: The typical example of these
• enzymes is amyloglucosidase or glucoamylase.
• This enzyme hydrolyzes α - D - (1-4) -D – glucosidic
bonds from the non-reducing ends to yield D – glucose
molecules.
• When the sequential removal of glucose reaches the
point of branching in amylopectin, the hydrolysis
continues on the (1-6) bonding but more slowly than
on the (1-4) bonding.
• Maltose is attacked only very slowly. The end product
is glucose.
• (iv) De-branching enzymes: At least two de-branching
enzymes are known: pullulanase and iso-amylase.
• Pullulanase: This is a de-branching enzyme which
causes the hydrolysis of α — D – (1 6) linkages in
amylopectin or in amylopectin previsouly attacked by
alphaamylase.
• It does not attack α - D (1-4) bonds. However, there
must be at least two glucose units in the group
attached to the rest of the molecules through an α -D(1-6) bonding.
• Iso-amylase: This is also a de-branching enzyme but
differs from pullulanase in that three glucose units in
the group must be attached to the rest of the
molecules through an α - D – (1 6) bonding for it to
function.
• (v) Enzymes that preferentially attack α - 1, 4 linkages:
Examples of this group are glucosidases.
• The maltodextrins and maltose produced by other
enzymes are cleaved to glucose by - glucosidases.
• They may however sometime attack unaltered
polysaccharides but only very slowly.
• (vi) Enzymes which hydrolyze starch to non-reducing
cyclic D-glucose polymers known as cyclodextrins or
Schardinger dextrins: Cyclic sugar residues are
produced by Bacillus macerans.
• They are not acted upon by most amylases although
enzymes in Takadiastase produced by Aspergillus
oryzae can degrade the residues.
• Industrial saccharification of starch by enzymes
• In industry the extent of the conversion of starch to
sugar is measured in terms of dextrose equivalent
(D.E.).
• This is a measure of the reducing sugar content,
determined under defined conditions involving
Fehling’s solution.
• The D.E is calculated as percentage of the total solids.
• Acid is being replaced more and more by enzymes.
• Sometimes acid is used initially and enzymes employed
later.
• Practical upper limit of acid saccharification is 55 D.E.
• Beyond this, breakdown products begin to accumulate.
• Furthermore, with acid hydrolysis reversion reactions
occur among the sugar produced.
• These two withdraws are avoided when enzymes are
utilized.
• By selecting enzymes specific sugars can be produced.
• Industrialy used enzymes are produced in
germinated seeds and by micro-organisms.
• Barley malt is widely used for the saccharification of
starch.
• It contains large amounts of various enzymes
notably -amylase and - glucosidase which further
split saccharides to glucose.
• All the enzymes discussed above are produced by
different micro-organisms and many of these
enzymes are available commercially.
• The most commonly encountered organisms
producing these enzymes are Bacillus spp,
Streptomyeces spp, Aspergillus spp, Penicillium spp,
Mucor spp and Rhizopus spp.
Cellulose, Hemi-celluloses and Lignin in
Plant Materials
• Cellulose
• Cellulose is the most abundant organic matter on
earth.
• Does not exist pure in nature and even the purest
natural form (that found in cotton fibres) contains
about 6% of other materials.
• Three major components, cellulose, hemi-cellulose
and lignin occur roughly in the ratio of 4:3:3 in
wood.
• Hemicelluloses
• Group of carbohydrates whose main and common
characteristic is that they are soluble in, and hence can
be extracted with, dilute alkali.
• They can then be precipitated with acid and ethanol.
• They are very easily hydrolyzed by chemically or
biologically.
• The nature of the hemicellulose varies among plants.
• In cotton the hemicelluloses are pectic substances,
which are polymers of galactose.
• In wood, they consist of short (DP less than 200)
branched heteropolymers of glucose, xylose, galactose,
mannose and arabinose as well as uronic acids of
glucose and galactose linked by 1 – 3, 1 – 6 and 1 – 4
glycosidic bonding.
• Lignin
• Lignin is a complex three-dimensional polymer
formed from cyclic alcohols. (Fig. 4.6).
• It is important because it protects cellulose from
hydrolysis.
• Cellulose is found in plant cell-walls which are held
together by a porous material known as middle
lamella.
• In wood the middle lamella is heavily impregnated
with lignin which is highly resistant and thus
protects the cell from attack by enzymes or acid.
• Pretreatment of cellulose-containing materials
before saccharification
• In order to expose lignocellulosics to attack, a
number of physical and chemical methods are in
use, or are being studied, for altering the fine
structure of cellulose and/or breaking the lignincarbohydrate complex.
• Chemical methods include the use of swelling
agents such a NaOH, some amines, concentrated
H2SO4 or HCI or proprietary cellulose solvents such
as ‘cadoxen’ (tris thylene-diamine cadmium
hydroxide).
• These agents introduce water between or within the
cellulose crystals making subsequent hydrolysis, easier.
• Steam has also been used as a swelling agent.
• The lignin may be removed by treatment with dilute
H2SO4 at high temperature.
• Physical methods of pretreatment include grinding,
irradiation and simply heating the wood.
• Hydrolysis of cellulose
• After pretreatment, wood may be hydrolyzed with
dilute HCI, H2SO4 or sulfites of Ca, Mg or Na under high
temperature and pressure.
• When, however, the aim is to hydrolyze wood to sugars,
the treatment is continued for longer than is done for
paper manufacture.
• Enzymatic hydrolysis has been subjected to many
research and work.
• Fungi was the main source of cellulolytic enzymes.
• Trichoderma viride and T. koningii have been the
most efficient cellulase producers.
• Penicillicum funiculosum and Fusarium solani have
also been shown to possess potent cellulases.
• Cellulase has been resolved into at least three
components: C1, Cx, and -glucosidases.
• The C1 component attacks crystalline cellulose and
loosens the cellulose chain, after which the other
enzymes can attack cellulose.
• Cx enzymes are β - (1 4) glucanases and hydrolyse soluble
derivatives of cellulose or swoollen or partially degraded
cellulose.
• Their attack on the cellulose molecule is random and
cellobiose (2-sugar) and cellotroise (3-sugar) are the major
products.
• Enzymes may also act by removing successive glucose units
from the end of a cellulose molecule.
• β-glucosidases hydrolyze cellobiose and short-chain oligosaccharides derived from cellulose to glucose, but do not
attack cellulose.
• They are able to attack cellobiose and cellotriose rapidly.
• Many organisms described in the literature as ‘cellulolytic’
produce only Cx and -glucosidases because they were isolated
initially using partially degraded cellulose.
• The four organisms mentioned above produce all three
members of the complex
• Molecular structure of cellulose
• Cellulose is a linear polymer of D-glucose linked in the
Beta-1, 4 glucosidic bondage.
• The bonding is theoretically as vulnerable to hydrolysis
as the one in starch.
• However, cellulose – containing materials such as wood
are difficult to hydrolyze because of:
• (a) the secondary and tertiary arrangement of cellulose
molecules which confers a high crystallinity on them
and
• (b) the presence of lignin.
• The degree of polymerization (D. P.) of cellulose
molecule is variable, but ranges from about 500 in
wood pulp to about 10,000 in native cellulose.
• When cellulose is hydrolyzed with acid, a portion
known as the amorphous portion which makes up
15% is easily and quickly hydrolyzed leaving a highly
crystalline residue (85%) whose DP is constant at
100-200.
• The crystalline portion occurs as small rod-like
particles which can be hydrolyzed only with strong
acid. (Fig. 4.7)